Increased expression of kisspeptin and GnRH forms in the brain of scombroid fish during final ovarian maturation and ovulation

ABSTRACT: BACKGROUND: Kisspeptins (Kiss) are prime players in the control of reproductive function through their regulation of gonadotropin-releasing hormone (GnRH) expression in the brain. The experimental scombroid fish, chub mackerel (Scomber japonicus) expresses two kiss (kiss1 and kiss2) and three gnrh (gnrh1, gnrh2, and gnrh3) forms in the brain. In the present study, we analyzed expression changes of kiss and gnrh mRNAs in the brain and corresponding GnRH peptides in the brain and pituitary during final ovarian maturation (FOM) and ovulation. METHODS: Female fish possessing late vitellogenic oocytes were injected with GnRH analogue to induce FOM and ovulation. Fish were observed for daily spawning activities and sampled one week post-injection at germinal vesicle migration (GVM), oocyte hydration, ovulation, and post-ovulatory time periods. Changes in relative mRNA levels of kiss and gnrh forms in the brain were determined using quantitative real-time PCR. Changes in GnRH peptides in the brain and pituitary were analyzed using time-resolved fluoroimmunoassay. RESULTS: Both kiss1 and kiss2 mRNA levels in the brain were low at late vitellogenic stage and increased significantly during the GVM period. However, kiss1 mRNA levels decreased during oocyte hydration before increasing again at ovulatory and post-ovulatory periods. In contrast, kiss2 mRNA levels decreased at ovulatory and post-ovulatory periods. Levels of gnrh1 mRNA in the brain increased only during post-ovulatory period. However, levels of gnrh2 and gnrh3 mRNAs were elevated during GVM and then, decreased during oocyte hydration before increasing again at ovulatory period. During post-ovulatory period, both gnrh2 and gnrh3 mRNA levels declined. Peptide levels of all three GnRH forms in the brain were elevated during GVM and oocyte hydration; their levels were significantly lower during late vitellogenic, ovulatory, and post-ovulatory periods. In contrast, pituitary GnRH peptide levels did not show any significant fluctuations, with the GnRH1 peptide levels being many-fold higher than the GnRH2 and GnRH3 forms. CONCLUSION: The results indicate increased expression of multiple Kiss and GnRH forms in the brain and suggest their possible involvement in the regulation of FOM and ovulation in captive female chub mackerel.     

Troponin is a potential regulator for actomyosin interactions

Troponin extracted from rabbit skeletal muscle directly binds to an actin filament in a molar ratio of 1:1 even in the absence of tropomyosin. An actin filament decorated with troponin did not exhibit significant difference from pure actin filaments in the maximum rate of actomyosin ATP hydrolysis and the sliding velocity of the filament examined by means of an in vitro motility assay. However, the relative number of troponin-bound actin filaments moving in the absence of calcium ions decreased to half that in their presence. The amount of HMM bound to the filaments was less than 4% of actin monomers in the presence of TNs. In addition, actin filaments could not move when Tn molecules were bound in the molar ratio of about 1:1 although they sufficiently bind to myosin heads. These results indicate that troponin can transform an actin monomer within a filament into an Off-state without sterically blocking of the myosin-binding sites with tropomyosin molecules.     

High-throughput transgenesis in Xenopus using I-SceI meganuclease

In this report we describe an easy, highly efficient transgenesis method for Xenopus. The method is very simple; a commercially available meganuclease, I-SceI, is incubated with a transgene construct carrying its recognition sites, and is subsequently microinjected into fertilized eggs. Approximately 30% (in Xenopus tropicalis) or 20% (in Xenopus laevis) of injected embryos exhibit non-mosaic, promoter-dependent transgene expression, and transgenes from the founder animals are transmitted to offspring. The method is compatible with mRNA or antisense morpholino oligonucleotide injection, and these secondary reagents can be introduced simultaneously or sequentially with a transgene to test their interaction. This high-throughput transgenic technique will be a powerful tool for studying the complex wiring of regulatory networks at the genome-wide level, as well as for facilitating genetic studies in the rapidly breeding diploid frog, X. tropicalis.     

Cloning and mapping of cat (Felis catus) immunoglobulin and T-cell receptor genes

 Molecular cloning and chromosomal mapping of the cat immunoglobulin (Ig) and T-cell receptor (TcR) genes were carried out to provide basic information for genetic analysis of immunologic diseases including leukemias and lymphomas in cats. We cloned two Ig constant genes, IGHM and IGHG and three TcR constant genes, TRAC, TRGC, and TRDC, by polymerase chain reaction (PCR) amplification of cDNA from cat peripheral blood mononuclear cells. For chromosomal mapping of the Ig and TcR loci including the IGK, IGL, and TRB on the cat genome, we performed PCR screening of DNAs from 37 cat × rodent somatic cell hybrids by using specific primers for the given genes. Consequently, three loci for IGH, TRA, and TRD, and two loci for TRB and TRG were found to be syntenic and assigned to cat chromosomes (FCA) B3 and A2, respectively. Further, IGK and IGL loci were mapped on FCA A3 and D3, respectively. These findings support the notion that the genetic linkages between the Ig and TcR genes are extensively conserved between humans and cats. Received: 18 June 1997 / Revised: 12 August 1997     

Genome-wide scan revealed that polymorphisms in the PNPLA3, SAMM50, and PARVB genes are associated with development and progression of nonalcoholic fatty liver disease in Japan

We examined the genetic background of nonalcoholic fatty liver disease (NAFLD) in the Japanese population, by performing a genome-wide association study (GWAS). For GWAS, 392 Japanese NAFLD subjects and 934 control individuals were analyzed. For replication studies, 172 NAFLD and 1,012 control subjects were monitored. After quality control, 261,540 single-nucleotide polymorphisms (SNPs) in autosomal chromosomes were analyzed using a trend test. Association analysis was also performed using multiple logistic regression analysis using genotypes, age, gender and body mass index (BMI) as independent variables. Multiple linear regression analyses were performed to evaluate allelic effect of significant SNPs on biochemical traits and histological parameters adjusted by age, gender, and BMI. Rs738409 in the PNPLA3 gene was most strongly associated with NAFLD after adjustment (P = 6.8 × 10^?14, OR = 2.05). Rs2896019, and rs381062 in the PNPLA3 gene, rs738491, rs3761472, and rs2143571 in the SAMM50 gene, rs6006473, rs5764455, and rs6006611 in the PARVB gene had also significant P values (<2.0 × 10^?10) and high odds ratios (1.84–2.02). These SNPs were found to be in the same linkage disequilibrium block and were associated with decreased serum triglycerides and increased aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in NAFLD patients. These SNPs were associated with steatosis grade and NAFLD activity score (NAS). Rs738409, rs2896019, rs738491, rs6006473, rs5764455, and rs6006611 were associated with fibrosis. Polymorphisms in the SAMM50 and PARVB genes in addition to those in the PNPLA3 gene were observed to be associated with the development and progression of NAFLD.     

Temocapril treatment ameliorates autoimmune myocarditis associated with enhanced cardiomyocyte thioredoxin expression

Thioredoxin (TRX) is a redox regulatory protein that protects cells from various stresses. Angiotensin-converting enzyme (ACE) inhibitor was reported to enhance endogenous antioxidant enzyme activities. This study was carried out to investigate whether temocapril, a novel non-sulfhydryl containing ACE inhibitor, reduces the severity of myocarditis via redox regulation mechanisms involving TRX. Western blot showed that temocapril enhanced cytosolic redox regulatory protein TRX expression, but neither mitochondrial TRX2 nor antioxidant enzymes, such as copper-zinc superoxide dismutase (Cu/Zn-SOD) or manganese superoxide dismutase (Mn-SOD) expression, was increased by the preconditioning treatment. In rats with experimental autoimmune myocarditis (EAM), the protein carbonyl content, a marker of cellular protein oxidation, was increased accompanied with enhanced TRX expression. An immunohistochemical study showed that TRX stain was enhanced in infiltrating inflammatory cells and in damaged myocytes. The severity of the myocarditis and the protein carbonyl contents were less increased in temocapril treatment (10 mg/kg/day, orally) from day 1 to day 21 in which TRX was up regulated when the inflammation started, but not in temocapril treatment from day 15–21 in which TRX was not up-regulated when the inflammation started. The results suggest that TRX and the redox state modified by TRX may play a crucial role in the pathophysiology of EAM. Temocapril ameliorates myocarditis associated with inducing TRX increase in a preconditioning manner, although the mechanism of TRX induction by temocapril remains to be elucidated.     

CYP98A6 from Lithospermum erythrorhizon encodes 4-coumaroyl-4'-hydroxyphenyllactic acid 3-hydroxylase involved in rosmarinic acid biosynthesis

Rosmarinic acid is the dominant hydroxycinnamic acid ester accumulated in Boraginaceae and Lamiaceae plants. A cytochrome P450 cDNA was isolated by differential display from cultured cells of Lithospermum erythrorhizon, and the gene product was designated CYP98A6 based on the deduced amino acid sequence. After expression in yeast, the P450 was shown to catalyze the 3-hydroxylation of 4-coumaroyl-4'-hydroxyphenyllactic acid, one of the final two steps leading to rosmarinic acid. The expression level of CYP98A6 is dramatically increased by addition of yeast extract or methyl jasmonate to L. erythrorhizon cells, and its expression pattern reflected the elicitor-induced change in rosmarinic acid production, indicating that CYP98A6 plays an important role in regulation of rosmarinic acid biosynthesis.     

Effects of interferon on the steroidogenic functions and proliferation of immature porcine granulosa cells in culture.

Recent studies suggest the relevance of several cytokines to the growth and differentiation of granulosa cells. In the present study, we investigated the effects of interferon (IFN) on the steroidogenic functions and proliferation of immature porcine granulosa cells. Human IFN-alpha inhibited FSH-induced progesterone secretion in a concentration-dependent manner. The effect of IFN-alpha was significant at a concentration as low as 10 pg/ml. Maximal inhibitory concentrations (10-50 ng/ml) of IFN-alpha reduced FSH-induced progesterone secretion by 70%. In contrast, estradiol secretion induced by FSH was significantly enhanced by relatively high concentrations (1-50 ng/ml) of IFN-alpha. IFN-alpha (0.1-10 ng/ml) reduced cAMP generation in response to FSH by as much as 80%, although its effect was not concentration-dependent. The proliferation of cultured granulosa cells was inhibited by IFN-alpha in a concentration-dependent manner. Human IFN-gamma did not affect granulosa cell functions. The stimulation of estradiol secretion and the inhibition of cell proliferation induced by IFN-alpha in cultured porcine granulosa cells in this study are in contrast with the effects of IL-1, which, as we reported previously, inhibited both progesterone and estradiol secretion and stimulated cell growth in these cell cultures. Such differences in the mode of action of cytokines may contribute to the regulation of granulosa cell functions under physiological or pathological conditions.